Anti-androgenic steroids

ABSTRACT

6 (H)-5,10-seco-4,5-dieneketo steroids are disclosed which exhibit anti-ardrogenic properties.

The invention described herein was made in the course of work under agrant or award from the Department of Health, Education and Welfare.

This is a division of application Ser. No. 661,792 filed Feb. 26, 1976now U.S. Pat. No. 4,059,630.

The present invention relates to certain new and useful steroids whichdemonstrate anti-androgenic properties. These steroids are5,10-secosteroids that have been found to be potent irreversibleinhibitors of the enzyme Δ⁵ -3-ketosteroid isomerase which is involvedin androgen biosynthesis. In addition, certain of the steroids of theinvention compete reversibly with 5-α-dihydrotestosterone for binding tothe prostatic cytoplasmic receptor protein while others inhibit theenzyme prostatic testosterone 5-α-reductase. The anti-androgenicproperties of the present compounds indicate such utility as the controlof abnormal growth of the human prostate, e.g. in the therapy of humanprostatic cancer.

BACKGROUND OF THE INVENTION

Prior studies¹ have shown that remarkably specific irreversible enzymeinhibitors can result from compounds bearing potential reactivegroupings which are unmasked at the active site by the target enzyme.This specificity resides in the generation of the alkylating agent bythe target enzyme at the active site as a result of the enzyme's normalcatalytic process. The process is exemplified by the enzymaticconversion of an acetylenic compound to an allene which can alkylate anactive site amino acid residue. The first such example was provided byBloch² who showed that the acetylenic analog of a normal substrate forβ-hydroxydecanoyl thioester dehydrase is converted by the enzyme to thecorresponding conjugated allenic thioester with rapid alkylation of anactive site histidine residue. This approach has been applied to theinhibition of monoamine oxidase³ and γ-cystathionase.⁴

The enzyme Δ⁵ -3-ketosteroid isomerase⁵ (EC 5.3.3.1) from Pseudomonastestosteroni converts C₁₉ and C₂₁ Δ⁵ -3-ketosteroids to thecorresponding Δ⁴ -3-ketosteroids. The proposed mechanism⁵,6 involvesremoval of the axial 4β-hydrogen with concomitant enolization to give aΔ³,5 -dienol, followed by ketonization with axial reprotonation at C-6.The hydrogen transfer from C-4 to C-6 is intramolecular. See Scheme 1below: ##STR1## The indicated reaction, when carried out by mammalian Δ⁵-3-ketosteroid isomerases is a key step in the biosynthesis of steroidhormones.

SUMMARY OF THE INVENTION

The present invention is based on the finding that there is rapidirreversible inhibition of bacterial Δ⁵ -3-ketosteroid isomerase bycertain novel acetylenic steroid analogs (illustrated by 1 and 2)(Scheme II). ##STR2## Compounds 1 and 2 above are, respectively,5,10-secoestr-5-yne-3,10,17-trione and5,10-seco-19-norpregn-5-yne-3,10,20-trione. For convenience, thesecompounds are referred to herein as compounds 1 and 2 or as "estryne" or"pregnyne", respectively.

It appears that the inhibition of bacterial Δ⁵ -3-ketosteroid isomeraseby compounds 1 and 2, or by other acetylenic steroids within the scopeof this invention, involves conversion of the β,γ-acetylenic ketone tothe conjugated allenic ketone via enzymatic enolization followed byketonization at C-3 with protonation at C-6. The conjugated allenicketones, which are also novel and constitute another embodiment of theinvention, then react readily with a nucleophilic residue at or near theactive site. This process finds analogy in studies withβ-hydroxydecanoyl thioester dehydrase.

While compounds (1) and (2) above (estryne and pregnyne, respectively)are preferred compounds, the invention is of broader scope and extendsto acetylenic steroids of the following structure A: ##STR3## wherein R¹stands for CH₂ =, O= or ##STR4## R² is O=, ##STR5## (where R³ is H orlower alkanoyl); ##STR6## (where R⁴ is H or lower alkanoyl); ##STR7##(where R⁵ is lower alkyl) or ##STR8## (where R⁶ is H, lower alkyl orCl); and R⁷ is H or CH₃.

Correspondingly, the allenic steroids of the invention are shown bystructures B and C: ##STR9## wherein R¹, R² and R⁷ have the meaningsgiven above.

As indicated, preferred compounds of structure A are5,10-secoestr-5-yne-3,10,17-trione and5,10-seco-19-norpregn-5-yne-3,10-20-trione, i.e. compounds 1 and 2,Scheme 2 above. These compounds may be synthesized by closely similarroutes. Thus, 3β,17β-diacetoxyestr-5(10)-en-6-one (3) may be prepared bydirect chromium trioxidepyridine oxidation of3β,17β-diacetoxyandrost-5-en-19-ol (cf. U.S. Pat. Nos. 3,159,621 and3,261,830). Epoxidation of (3) with m-chloroperbenzoic acid in benzene(reflux) or by reaction with alkaline hydrogen peroxide followed byreacetylation gives the corresponding 5β-10β-oxidosteroid (4), mp245°-247°. Fragmentation of (4) with p-toluene sulfonylhydrazine^(6') atambient temperature in acetic acid-chloroform (1:1) gives3β,17β-diacetoxy-5,10-secoestr-5-yn-10-one (5), mp 204°-206°, in highyield. Hydrolysis of the acetate groups in (5) (3% methanolic KOH)followed by oxidation with Jones reagent⁷ gives (1). Compound (2) may besynthesized by exactly the same sequence of reactions starting with3β,20-diacetoxy-5-pregnen-19-ol.

Incubation of crystalline Δ⁵ -ketosteroid isomerase at pH 7.0 withacetylenic steroids (1) and (2) in 1,4-dioxane results in rapidirreversible and complete inactivation of the enzyme. The inactivationis progressive with time, and half-lives in the range of 150-1320 secare observed at concentrations of 20-200 μM of the two inhibitors. Theenzymatic activities of control vessels which received only equivalentvolumes of 1,4-dioxane remained constant at initial activities. Evidencefor the irreversible nature of the inhibition is based on: inability torestore enzymatic activity by prolonged dialysis (24 hr at 4° vs. 1 mMpotassium phosphate buffer, pH 7.0); the fact that extensively dilutedpartially inhibited enzyme preparations retain constant activity formany days; and the kinetic behavior described below.

The initial rates of inactivation of the isomerase by (1) and (2) can beanalyzed by the method of Kitz and Wilson⁸ since very satisfactorypseudo-first-order behavior was observed. It is assumed that [I](inhibitor) >> [E] (enzyme), that [E.I] (the reversible enzyme-inhibitorcomplex) is at all times in equilibrium with enzyme and inhibitor, andthat ^(k) cat >> ^(k) inh. The scheme for the formation of EI' (theirreversible enzyme-inhibitor derivative) may then be represented asfollows.

If the enzyme-inhibitor solution is diluted extensively prior to assay,the active enzyme (ε) = [E] + [E.I]. Then -d(ε)/dt = k₃ [E.I.], and K₁ =([E][I])/[EI]. Thus ##EQU2## where E_(t) = total amount of enzyme insystem.

If one defines ##EQU3## Consequently double reciprocal plots of k_(app)with respect to [I] should be linear, with slopes and interceptspermitting the determination of K₁ and k₃ where the latter is theoverall rate constant for the irreversible inhibition process.

From plots of 1n ε (residual enzymatic activity) vs. time, k_(app) hasbeen determined at five inhibitor concentrations for both compounds 1and 2, i.e. estryne and pregnyne. Strict linearity of thesemilogarithmic plots has been observed in all cases, over greater thantwo half-lives. A plot of 1/k_(app) vs. 1/[I] was linear and gave k₃ andK₁ (FIG. 1). For compound 1, K₁ = 56 μM and k₃ = 1.98 × 10⁻³ sec⁻¹, andfor compound 2, K₁ = 32 μM and k₃ = 4.10 × 10⁻³ sec⁻¹.

These experiments indicate that the acetylenic steroids (1) and (2)inactivate Δ⁵ -3-ketosteroid isomerase by covalent linkage to theenzyme. The inactivation is rapid and specific, presumably because theisomerase enzyme generates the alkylating system at its active site byexercising its normal catalytic function.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The following examples illustrate the preparation of the preferredcompounds (1) and (2) above.

EXAMPLE 1

3β,17β-diacetoxyestr-5(10)-en-6-one

A solution of 3β,17β-diacetoxy-19-hydroxy androst-5-ene (20g) inpyridine (80ml) is added in one portion to a stirred mixture of chromiumtrioxide (40g) and pyridine (400ml) at 25°. The reaction mixture isstirred at 25° for 96 hours, and is then diluted with ether (1200ml) andthen is filtered through Celite. The residue on the filter is washedwith ether and the combined filtrate and washings are washedsuccessively with 5% aqueous sodium hydroxide, water, 10% aqueoushydrochloric acid, water, then dried (Na₂ SO₄) and evaporated todryness. The residue is subjected to dry column chromatography on silicagel, eluting with chloroform-ethyl acetate (15:1) and there results thecompound of this Example (1), mp 123°-124° (after crystallization fromacetone-petroleum ether; mass spectrum m/e 374 (M⁺), 332, 314, 286.

EXAMPLE 2

3β,17β-diacetoxy-5β,10β-oxidoestran-6-one

To a solution of 3β,17β-diacetoxyestr-5(10)-en-6-one (10.2g) in benzene(820ml) is added m-chloro perbenzoic acid (15.7g) and the solution isheated under reflux for 1 hour. The reaction mixture is then cooled,washed successively with water, 5% aqueous sodium bicarbonate solutionand water, is dried (Na₂ SO₄) and evaporated in vacuo to give a solidresidue. Crystallization from methylene chloride-acetone gives thecompound of this Example (2) mp 245°-247°; mass spectrum, m/e 390 (M⁺),330, 314, 302, 286.

EXAMPLE 3

3β,17β-diacetoxy-5,10-secoestr-5-yn-10-one

To a stirred solution of 3β,17β-diacetoxy-5β,10β-oxidoestran-6-one(6.0g) in a mixture of chloroform (100ml) at 25° is added p-toluenesulfonyl hydrazine (1.24g). Stirring is continued for 6 hours at 25°,and the reaction mixture is then diluted with water and extracted withchloroform. The chloroform extract is then washed successively withwater, 5% aqueous sodium bicarbonate solution and water, and is dried(Na₂ SO₄) and evaporated in vacuo to give a solid residue. This residueis crystallized from methanol to give the compound of this Example (3)(1.7g) as plates mp 204°-206°; mass spectrum m/e 374 (M⁺), 332, 316,304, 286.

EXAMPLE 4

3β,17β-dihydroxy-5,10-secoestr-5-yn-10-one

3β,17β-dihydroxy-5,10-secoestr-5-yn-10-one (374mg) is dissolved in 3%methanolic potassium hydroxide (75ml) and the solution is stirred at 25°for 3 hours. The reaction mixture is then evaporated in vacuo at 30° toabout 10ml, and saturated aqueous sodium chloride solution (40ml) isadded. The resulting mixture is extracted with chloroform, and thechloroform extract is washed with water, dried (Na₂ SO₄) and evaporatedin vacuo to give the compound of this Example (4), mp 207°-208°; massspectrum m/e 290 (M⁺), 272, 262, 254, 244.

EXAMPLE 5

5,10-secoestr-5-yne-3,10,17-trione ("Estryne")

To a solution of 3β,17β-dihydroxy-5,10-secoestr-5-yn-10-one (4; 1.0g) inacetone (150ml) at 5° is added, dropwise, Jones' reagent (chromiumtrioxide in sulfuric acid-water) until a permanent orange-browncoloration persists. After 5 minutes at 5°, the mixture is treated withmethanol to destroy excess chromic acid and is diluted with water andextracted with ether. The ethereal extract is washed with water, dried(Na₂ SO₄) and evaporated to give a solid residue. Crystallization fromacetone-petroleum ether gives the compound of this Example (5), mp142°-145°; mass spectrum m/e 286 (M⁺), 271, 258, 243, 230, 215.

EXAMPLE 6

3β,20β-diacetoxy-19-norpregn-5(10)-en-6-one

A solution of 3β,20β-diacetoxy-19-hydroxypregn-5-ene (32g) in pyridine(120ml) is added in one portion to a stirred mixture of chromiumtrioxide (60g) and pyridine (600ml) at 25°. The reaction mixture isstirred at 25° for 96 hours and is diluted with ether (1800ml) and thenis filtered through Celite. The residue on the filter is washed withether, and the combined filtrate and washings are washed successivelywith 5% aqueous sodium hydroxide, water, 10% aqueous hydrochloric acid,water, then dried (Na₂ SO₄) and evaporated to dryness. The residue issubjected to dry column chromatography or silica gel, eluting withchloroform-ethyl acetate (15:1) and there results the compound of thisExample (6), mp 144°-148°; λ_(max) ^(MeOH) 248 nm (E=10,400).

EXAMPLE 7

3β,20β-diacetoxy-5β,10β-oxido-19-norpregnan-6-one

To a solution of 3β,20β-diacetoxy-19-norpregn-5(10)-en-6-one (5g) inbenzene (400ml) is added m-chloroperbenzoic acid (8g) and the solutionis heated under reflux for 1 hour. The reaction mixture is then cooled,washed successively with water, 5% aqueous sodium bicarbonate solutionand water, is dried (Na₂ SO₄) and evaporated in vacuo to give a solidresidue. Crystallization from hexane-ethanol gives the compound of thisExample (7), mp. 134°-136°; mass spectrum, m/e 418, 360, 358, 342, 340,330.

EXAMPLE 8

3β,20β-diacetoxy-5,10-seco-19-norpregn-5-yn-10-one

To a stirred solution of3β,20β-diacetoxy-5β,10β-oxido-19-norpregnan-6-one (9g) in a mixture ofchloroform (150ml) and glacial acetic acid (150ml) at 25° is addedp-toluene sulfonyl hydrazine (1.9g). Stirring is continued for 6 hoursat 25°, and the reaction mixture is then diluted with water andextracted with chloroform. The chloroform extract is then washedsuccessively with water, 5% aqueous sodium bicarbonate solution andwater, and is dried (Na₂ SO₄) and evaporated in vacuo to give a solidresidue. This residue is crystallized from acetone-hexane to give thecompound of this Example (8), mp. 132°-134°; mass spectrum m/e 402 (M⁺),360, 342, 282.

EXAMPLE 9

3β,20β-dihydroxy-5,10-seco-19-norpregn-5-yn-10-one

3β,20β-diacetoxy-5,10-secoestr-5-yn-10-one (1.0g) is dissolved in 3%methanolic potassium hydroxide (150ml) and the solution is heated underreflux for 3 hours. The reaction mixture is cooled, evaporated in vacuoat 30° to about 20 ml and saturated aqueous sodium chloride solution(80ml) is added. The resulting mixture is extracted with chloroform, andthe chloroform extract is washed with water, dried (Na₂ SO₄) andevaporated in vacuo to give the compound of this Example (9), mp.187°-188°; mass spectrum m/e 318 (M⁺), 300, 285, 282, 272.

EXAMPLE 10

5,10-seco-19-norpregn-5-yne-3,10,20-trione ("Pregnyne")

To a solution of 3β,20β-dihydroxy-5,10-seco-19-norpregn-5-yn-10-one(1.0g) in acetone (150ml) at 5° is added, dropwise, Jones' reagent(chromium trioxide in sulfuric acid-water) until a permanentorange-brown coloration persists. After 5 minutes at 5° methanol isadded to the mixture to destroy excess chromic acid, and the mixture isthen diluted with water and extracted with ether. The ethereal extractis washed with water, dried (Na₂ SO₄) and evaporated to give a solidresidue. Crystallization from acetone-petroleum ether gives the compoundof this Example (10), mp. 156°-159°; mass spectrum m/e 314 (M⁺), 286,271.

Other examples include 5,10-seco-androst-5-yn-10(19)-ene-3,17-dione and5,10-seco-pregn-5-yn-10(19)-ene-3,20-dione. The synthetic routes tothese compounds are exactly analogous to each other and are illustrativeof the preparation of those compounds of structure A, where R¹ = CH₂.

EXAMPLE 11

19-p-Toluenesulfonyloxy-3β,17β-diacetoxyandrost-5-ene

To a solution of 3β,17β-diacetoxy-19-hydroxyandrost-5-ene (14.4g) inpyridine (370ml) at 25° is added p-toluenesulfonyl chloride (38.1g) andthe solution is left at 25° for 4 days. Water is then added, and themixture is extracted with ethyl acetate, and the organic extract iswashed successively with water, 5% aqueous hydrochloric acid, 5% aqueoussodium bicarbonate and water, and then dried (Na₂ SO₄) and evaporated invacuo. The residue is crystallized from methylene chloride-hexane togive the compound of this Example (11), mp. 135°-137°; mass spectrum m/e484, 372, 312.

EXAMPLE 12

3β,17β-diacetoxy-6β-hydroxy-5β,19-cycloandrostane

A solution of 19-p-toluenesulfonyloxy-3β,17β-diacetoxy androst-5-ene(15.0g) and potassium acetate (11.76g) in acetone-water (3:1; 750ml) isheated under reflux for 48 hours. The acetone is then removed in vacuoand the resulting aqueous mixture is extracted with ethyl acetate. Theorganic extract is washed with water, dried (Na₂ SO₄) and evaporated invacuo to give the crude product which is chromatographed on silica gel(eluant: chloroform-ethyl acetate, 9:1) to give the compound of thisExample (12) as an oil, mass spectrum m/e 390(M⁺), 372, 330, 312, 270.

EXAMPLE 13

3β,17β-diacetoxy-5β,19-cycloandrostan-6-one

A solution of 3β,17β-diacetoxy-6β-hydroxy-5β,19-cycloandrostane (1.3g)in acetone (100ml) at 5° is treated with Jones' reagent (CrO₃ --H₂ SO₄)until a permanent brown coloration persists. After 5 minutes at 5°, themixture is concentrated in vacuo to about 20ml and is diluted withwater. The aqueous mixture is extracted with ethyl acetate, washed withwater, dried (Na₂ SO₄) and evaporated in vacuo to give a solid residue.Crystallization from methylene chloride-hexane gives the compound ofthis Example (13), mp. 151°-152°, mass spectrum m/e 328, 313, 300, 268,253, 225.

EXAMPLE 14

3β,17β-diacetoxy-5β,19-cycloandrostan-6-one-p-toluenesulfonylhydrazine

To a solution of p-toluenesulfonylhydrazine (205mg) is methanol (10ml)is added 3β,17β-diacetoxy-5β,19-cycloandrostan-6-one (388mg) and themixture is left at 25° for 24 hours. The reaction mixture is thenfiltered, and the residue on the filter is the pure compound of thisExample (14), mp. 288°-290°.

EXAMPLE 15

3β,17β-diacetoxy-5,10-secoandrost-5-yn-10(19)-ene

A mixture of 3β,17β-diacetoxy-5β,19-cycloandrostan-6-onep-toluenesulfonylhydrazine (2.78g) and sodium hydride (150mg; added asdispersion in mineral oil) is suspended in o-xylene (200ml), and heatedunder reflux for 3 hours. The reaction mixture is then cooled, washedsuccessively with 5% aqueous sodium bicarbonate and water, dried (Na₂SO₄) and evaporated to give a residue comprising substantially thecompound of this Example (15), mass spectrum m/e 372(M⁺), 330, 312, 297,252.

EXAMPLE 16

5,10-secoandrost-5-yn-10(19)-ene-3,17-dione

The product of Example (15) is converted to a mixture containing thecompound of this Example (16) by basic hydrolysis to give the3β,17β-diol and oxidation of the 3β,17β-diol by Jones' reagent,according to the procedures of Examples (4) and (5) above.

EXAMPLE 17

The enzyme inhibitory properties of the compounds of this invention areillustrated by the following data obtained with the preferred compoundsof the invention, i.e. compounds (1) and (2) (i.e. the "Estryne" and"Pregnyne" products of Examples (5) and (10), respectively).

Studies were performed using crystalline purified bacterial Δ⁵-3-ketosteroid isomerase (from P. testosteroni), under pseudo firstorder conditions using the kinetic analysis of R. Kitz and I. B. Wilson[J. Biol. Chem., 237, 3245 (1964)]. Powerful irreversible inhibition ofthe enzyme was demonstrated thereby, with K_(I) = 56μM and k₃ = 1.98 ×10⁻³ sec.⁻¹ for compound (1) and K_(I) = 32 μM and k₃ = 4.1 × 10⁻³sec.⁻¹ for compound (2) (10-fold range of inhibitor concentrations).

In Vivo Properties

Studies with intact male Sprague-Dawley rats (300g body weight) areillustrated for compounds (1) and (2). Thus, subcutaneous administrationin sesame oil of 13mg per kilo per day of compound 5 for 7 days resultedin 30% reduction in weight of the ventral prostate. Furthermore,administration of compound (1) in propylene glycol intraperitoneally ata dosage of 20mg per kilo per day for 7 days resulted in 28% ofreduction in weight of the ventral prostate.

    ______________________________________                                                             Average Weight of                                        Compound and Dosage  Ventral Prostate                                         ______________________________________                                        Controls; 0.2ml sesame oil                                                                         238 mg                                                   per rat per day for 7 days, s.c.                                              Compound 5,                                                                   13mg/Kg in sesame oil (0.2ml)                                                                      167 mg                                                   per rat per day for 7 days, s.c.                                              Controls; 0.2ml propylene glycol                                                                   421 mg                                                   per rat per day for 7 days, i.p.                                              Compound 5; 13mg/Kg in propylene                                              glycol (0.2ml)       302 mg                                                   per rat per day for 7 days, i.p.                                              ______________________________________                                    

Similarly, compound (2), when administered in propylene glycolintraperitoneally to intact male Sprague-Dawley rats at a dosage of 20mgper kilo for 7 days produced 20% reduction in weight of the ventralprostate and 28% reduction of the dorsal lateral prostate.

The following Chart I illustrates a synthetic route for the preparationof compounds 1(a-c) according to the invention: ##STR10##

The critical part of the process route involves generation of the keyΔ⁵(10) -6-oxo intermediate (3a) with subsequent fragmentation of thederived 5β,10β-oxido-6-ketone (4a) to (5a) by the Tanabe-Eschenmoser⁹procedure. The compound (3a) is prepared by direct conversion of Δ⁵-19-hydroxysteroids to Δ⁵(10) -6-ketones using chromiumtrioxide-pyridine at 25°. (See U.S. Pat. Nos. 3,159,621 and 3,261,830).This method (4 days at ambient temperature followed by chromatrographyon silica gel) gives the Δ⁵(10 -6-ketone (3a) conveniently in 40% yield.Epoxidation of 3a to give the oxidoketone (4a) is carried out usingalkaline hydrogen peroxide, followed by reacetylation at C-3 for easierisolation and characterization.

The 5β,10β-configuration for 4a is inferred from the negative c.d.curve,¹⁰ as well as from the following evidence:

The known 3β-acetoxy-Δ⁵(10) -6β-ol (7) of established¹¹ stereochemistryis converted quantitatively by m-chloroperbenzoic acid to the 5,10-oxidocompound (8). The well established¹² directive effect of the hydroxylgroup in the peracid epoxidation of allylic alcohols indicates the5β,10β-configuration of the epoxide grouping in 8. Oxidation of 8 withJones reagent then gives in quantitative yield, the oxidoketone (4a),identical in all respects with 4a prepared by H₂ O₂ --NaOH epoxidationof the conjugated ketone 3a followed by reacetylation at C-3.

The 5β,10β-oxido-6-ketone 4a is then fragmented to give 5a by theTanabe-Eschenmoser reaction (p-toluenesulfonylhydrazide in aceticacid-chloroform at room temperature). Spectroscopic and analytical dataare consistent with structure 5a while positive evidence for theacetylenic grouping from the Raman spectrum which showed absorption at2230 cm⁻¹. In addition, catalytic hydrogenation of 5a using Adamscatalyst gives the tetrahydro derivative.

The 3β-acetoxy-5,10-secosteroid 5a is then hydrolyzed to the 3β-ol (6a),and oxidation with Jones reagent then affords, in ca. 50% yield, thefinal product 1a, which gives appropriate spectroscopic and analyticaldata.

The marked lack of reactivity of the C-10 carbonyl group in 5a isnoteworthy. Attempts to prepare the p-toluenesulfonylhydrazone havefiled, even under forcing conditions (e.g. p-toluenesulfonylhydrazideand p-toluenesulfonic acid in sulfolanedimethylformamide at 100°).Attempts to generate the oxime, using conditions(hydroxylamine-pyridine, reflux) suitable¹³ for hindered 11-oxosteroidshave also failed. Furthermore, no reduction of the 10-ketone has beenobserved under forcing Wolff-Kishner conditions, or by the use of sodiumborohydride or lithium aluminum hydride. This lack of reactivity mightbe due in part to electronic interaction between the acetylene and10-carbonyl functions. However, this is unlikely to be a major factor,as the tetrahydro compound obtained by catalytic hydrogenation of theacetylenic compound 5a has also proven to be inert to hydrazone-formingreagents. The formation of an intermediate complex would result insevere transannular interactions in the 10-membered ring, and thiseffect may play a critical role.

The sequence of Chart I as outlined above for the cholestane series canbe applied in the preparation of the 5,10-secoestryne (1b) and5,10-seco-19-norpregnyne (1c). It has been observed that the5β,10β-oxidoketone system (e.g. 4b) can be conveniently prepared bytreatment of the corresponding Δ⁵(10) -6-ketone with m-chloroperbenzoicacid in benzene under reflux. The undesired Baeyer-Villiger reactiondoes not compete to a major extent, and this procedure gives thecrystalline oxidoketone 4b directly in 67% yield without thereacetylation required after the H₂ O₂ --NaOH procedure.

The 5β,10β-configuration for the oxido compounds 4b and 4c is inferredfrom their negative c.d. curves, as well as from analogy with thecholestane series. In addition, chromous acetate¹⁴ reduction of 4b givesthe 10β-hydroxy-6-ketone 9, which shows a negative c.d. spectrum asexpected for a 6-oxosteroid of the 5α,10β-series.

The final products 1b and 1c are obtained by fragmentation ofoxidoketones 4b and 4c, followed by hydrolysis of the acetate groupingsand oxidation with Jones reagent, as for the cholestane series. Itshould be noted that hydrolysis of the acetoxy groups in 5 a-c with basedoes not cause epimerization at C-9. Thus, reacetylation of thehydrolyzed products 6 a-c generates, in quantitative yield, unchanged 5a-c as evidenced by spectroscopic, chromatographic and c.d.measurements.

The following additional examples further illustrate the invention:

EXAMPLE 18

3β-acetoxy-19-norcholest-5(10)-en-6-one(3a)

3β-acetoxy-19-hydroxycholest-5-ene(15.8g, 0.036 mole) was dissolved inpyridine (100ml) and stirred with chromium trioxide-pyridine complex(0.30 mole) at ambient temperature for 4 days. The mixture was dilutedwith EtOEt(1.5l) and filtered. The ethereal phase was washedsuccessively with 5% NaOH and H₂ O. The residue obtained byconcentration in vacuo was chromatographed on silica gel by elution with6% EtOAc--CHCl₃. Early fractions contained several non-polar compoundsfollowed by (3a) (5.7g, 0.0133 mole, 36%) mp 122°-123° (from MeOH) massspectrum m/e 428(M⁺), 400, 382, 368; λ_(max) ^(MeOH) 246nm (ε11,430);[θ] (MeOH) +2530° (329nm);νmax(KBr)1725 (ester C═O), 1660(C═O) 1660cm⁻¹(C═C); nmr (C₆ D₆)4.50(m,1,CHOAc), 2.46(m,2,COCH₂), 2.00(s,3,CH₃ CO),0.85(s,3,18 -CH₃)

Anal. Calcd. for C₂₈ H₄₄ O₃ : C,78.45; H,10.35. Found: C,78.43; H,10.15.

EXAMPLE 19

3β-acetoxy-5β,10β-oxido-19-norcholestan-6-one (4a)

3β-Acetoxy-19-norcholest-5(10)-en-6-one (3a, 235mg, 0.00055 mole) wasdissolved in MeOH--CHCl₃ (15ml: 3ml). At ambient temperature a mixtureof 30% H₂ O₂ (1ml) and 5N NaOH (1ml) was added. After stirring for 5 hrthe reaction mixture was diluted with aqueous NaCl(sat) and extractedwith CHCl₃. Drying (Na₂ SO₄) and removal of solvent in vacuo left asolid which was acetylated with acetic anhydride-pyridine (18 hr., roomtemperature). After work up in the usual manner the residue wascrystallized from MeOH to give 4a as needles (150mg, 0.00033 mole, 58%):mp 173°-174°; mass spectrum m/e 444 (M⁺), 384, 368, 356, 340;νmax 1735(ester C═O), 1705cm⁻¹ (C═O); [θ] (CH₃ OH) - 8894° (307.5nm); nmr 4.62(m, 1,CHOAc), 2.02 (s,3,CH₃ CO), 0.70 (s,3,18-CH₃)

Anal. Calcd. for C₂₈ H₄₄ O₄ : C,75.63; H,9.97. Found: C,75.48; H,9.79.

EXAMPLE 20

3β-acetoxy-5β,10β-oxido-19-norcholestan-6-one (4a) from3β-Acetoxy-6βhydroxy-19-norcholest-5(10)-ene (7)

A solution of the Δ⁵(10) -6β-ol¹¹ (7, 54mg, 0.00013 mole) and 85%m-chloroperbenzoic acid (54mg, 0.00027 mole) in CHCl₃ (8ml) was left atambient temperature for 18 hr. Water was then added, and the CHCl₃ phasewas washed successively with 10% aqueous Na₂ SO₃ solution, 10% NaHCO₃solution and water, and then was dried (Na₂ SO₄) and evaporated. Theresidue was crystallized from acetone to give 8, mp 140°-141°; massspectrum m/e 428 (M-18), 386, 368.

Oxidation of the above product with Jones reagent in acetone at 5° for15 minutes gave crude 4a as a crystalline product. This material washomogenous by tlc, had mp 173°-174° and was identical in all respects(ir, nmr, cd, ms) with a sample of 4a, prepared by H₂ O₂ --NaOHepoxidation of the conjugated ketone 3a followed by reacetylation atC-3.

EXAMPLE 21

3β-acetoxy-5,10-seco-19-norcholest-5-yn-10-one (5a)

3β-Acetoxy-5,10-oxidocholestan-6-one (4a, 666mg, 0.0015 mole) andp-toluenesulfonylhydrazide (333mg, 0.0018 mole) were dissolved in 1:1CHCl₃ --AcOH (50ml). After stirring 5 hr at ambient temperature thereaction mixture was diluted with water and CHCl₃. The organic phase waswashed with water and 5% NaHCO₃. Removal of the dried (Na₂ SO₄) solventin vacuo gave an oil which crystallized from MeOH, giving 5a as plates(471mg, 0.0011 mole, 73%): mp 105°-106°; mass spectrum m/e 428 (M⁺),386, 368, 350; νmax 1730 (ester C═O), 1705cm⁻¹ (C═O); [θ] (CH₃ OH) -2805° (283nm); nmr 4.80 (m,1,CHOAc), 2.02 (s,3,CH₃ CO), 0.76(s,3,18-CH₃)

Anal. Calcd. for C₂₈ H₄₄ O₃ : C,78.45; H,10,35; O,11.20. Found: C,78.27;H,10.35; O,11.20.

EXAMPLE 22

3β-hydroxy-5,10-seco-19-norcholest-5-yn-10-one (6a)

The acetate (5a) (35mg, 0.00008 mole) was dissolved in MeOH (7ml) andstirred with anhydrous K₂ CO₃ (150mg) for 2 hr. Filtration andevaporation of the solvent left a solid which was crystallized fromhexane to give 6a as needles (30mg, 0.000078 mole, 97%): mp 153°-154°;mass spectrum m/e 386 (M⁺), 368, 340; νmax (KBr) 3400 (OH), 1705cm⁻¹(C═O); [θ] (CH₃ OH) - 2556° (283nm); nmr 3.84 (m,1,CHOH), 0.75(s,3,18-CH₃).

Anal. Calcd. for C₂₆ H₄₂ O₂ : C,80.83; H,10.88. Found: C,80.66; H,10.79.

EXAMPLE 23

5,10-seco-19-norcholest-5-yne-3,10-dione (1a)

3β-Hydroxy-5,10-Seco-19-norcholest-5-yn-10-one (6a, 163mg, 0.00042 mole)was dissolved in acetone (50ml) and oxidized with excess Jones reagentfor 10 minutes at ambient temperature. The mixture was diluted with H₂ Oand extracted with CHCl₃. Drying (Na₂ SO₄) and removal of the solvent invacuo gave an oil which was crystallized from MeOH-EtOEt to give (1a,77mg, 0.0002 mole): mp 94°-96°; mass spectrum m/e 384 (M⁺), 269, 256,299; [θ] (dioxane) - 6696° (287nm); νmax (KBr) 1705cm⁻¹ ; nmr 0.75(s,3,18-CH₃).

Anal. Calcd. for C₂₆ H₄₀ O₂ : C,81.25; H,10.42. Found: C,81.49; H,10.53.

EXAMPLE 24

3β-17β-diacetoxyestr-5(10)-en-6-one (3b)

Compound 3b was prepared in a manner identical with that for (3a) asdescribed above: mp 116°-118°; mass spectrum m/e 374 (M⁺), 332, 314,286; λ_(max) ^(MeOH) 245nm (ε11,078); [θ] (CH₃ OH) +2372° (330nm); νmax(KBr) 1730 (ester C═O), 1660 (C═O), 1620cm⁻¹ (C═C); nmr 5.11(m,1,CHOAc), 4.66 (m,1,CHOAc), 2.02 (s,3,CH₃ CO), 2.00 (s,3,CH₃ CO),0.86 (s,3,18-CH₃)

Anal. Calcd. for C₂₂ H₃₀ O₅ : C,70.58; H,8.02. Found: C,70.31; H,8.14.

EXAMPLE 25

3β,17β-diacetoxy-5β,10β-oxidoestran-6-one (4b)

A mixture of 3β,17β-Diacetoxyestr-5(10)-en-6-one (3b, 3.74g, 0.01 mole)and 85% m-chloroperbenzoic acid (5.74g, 0.028 mole) was refluxed inbenzene (300ml) for 1 hr. The cooled solution was washed successivelywith water, 5% NaHCO₃, water and dried (Na₂ SO₄). The residuecrystallized from MeOH, after removal of the solvent in vacuo, to givepure epoxide (4b). Chromatography of the mother liquor on silica gel(elution with 8% EtOAc-CHCl₃) gave additional (4b), total yield 2.7g0.0067 mole, 67%): mp 245°-247°; mass spectrum m/e 390 (M⁺), 330, 314,302, 286; νmax 1725 (ester C═O), 1705 (C═O), 1250cm⁻¹ (ester); [θ] (CH₃OH) -8506° (306nm); nmr (C₆ D₆) 4.60 (m,2,CHOAc), 2.06 (s, 3,CH₃ CO)2.01 (s,3,CH₃ CO), 0.84 (s,3,18-CH₃)

Anal. Calcd. for C₂₂ H₃₀ O₆ : C,67.67; H,7.74. Found: C,67.65; H,7.66.

EXAMPLE 26

3β,17β-diacetoxy-5β,10β-oxidoestran-6-one (4b) from3β,6β,17β-Triacetoxyestr-5(10)-ene (10)

To a solution of the triacetate (10, 1.0g, 0.0026 mole) in acetone(40ml) at 25° was added Jones reagent (1.2ml) with swirling. After 10min, excess reagent was destroyed by dropwise addition of MeOH, and thereaction mixture was diluted with water, and extracted with CHCl₃. TheCHCl₃ extract was washed with water, dried (Na₂ SO₄) and evaporated invacuo to give a solid residue which was chromatographed on silica gel(elution with CHCl₃ -EtOAc, 9:1). The first fractions contained startingmaterial (10, 119mg), and later fractions contained the oxidoketone (4b,200mg), mp 247° (from MeOH) identical in all respects (ir, nmr, tlc, ms)with an authentic sample of 4b.

EXAMPLE 27

3β,17β-diacetoxy-5,10-secoestr-5-yn-10-one (5b)

A mixture of3β,17β-Diacetoxy-5β,10β-oxidoestran-6-one (4b, 2.0g, 0.005mole) and p-toluenesulfonylhydrazide (1.24g, 0.0067 mole) was stirredfor 6 hrs. in 1:1 CHCl₃ --AcOH (150ml) at ambient temperature. The CHCl₃extract, after dilution with water, was washed with 5% NaHCO₃, water anddried (Na₂ SO₄). Removal of the solvent in vacuo and crystallization ofthe residue (MeOH) gave 5b as plates (1.7g, 0.0045 mole, 90%): mp204°-206°; mass spectrum m/e 374 (M⁺), 332, 316, 304, 286; max 1724(C═O), 1250cm⁻¹ (ester); [θ] (CH₃ OH) -2710° (284nm); nmr 4.62(m,2,CHOAc), 2.01 (s,6,CH₃ CO at C-3 and C-17), 0.88 (s,3,18-CH₃).

Anal. Calcd. for C₂₂ H₃₀ O₅ : C,70.56; H,8.08. Found: C,70.70; H,7.94.

EXAMPLE 28

3β,17β-dihydroxy-5,10-secoestr-5-yn-10-one (6b)

The diacetate (5b, 374mg, 0.001 mole) was stirred at ambient temperaturefor 3 hr in 3% methanolic KOH (75ml). Concentration in vacuo after theaddition of sat. NaCl (40ml), extraction with CHCl₃, drying (Na₂ SO₄)and removal of the solvent gave the pure diol (6b). Crystallization fromhexane-EtOH gave 6b as needles (280mg, 0.0096 mole, 96%): mp 205°-207°;mass spectrum m/e 290 (M⁺), 272, 262, 254, 244; νmax (KBr) 3440 (OH),1725cm⁻¹ (C═O); [θ] (CH₃ OH) -2744° (283nm); nmr (d₆ -DMSO) 4.85(m,1,CHOH), 4.57 (m,1,CHOH), 0.78 (s,3,18-CH₃).

Anal. Calcd. for C₁₈ H₂₆ O₃ : C,74.44; H,9.03. Found: C,74.21; H,8.99.

EXAMPLE 29

5,10-secoestr-5-yne-3,10,17-trione (1b)

The 3β,17β-diol (6b, 186mg, 0.0006 mole) was dissolved in acetone (40ml)and oxidized with excess Jones reagent for 5 min at ambient temperature.The reaction was diluted with water and extracted with CHCl₃. Theresidue obtained after removal of solvent was crystallized frompetroleum ether --CHCl₃ to give 1b as plates (98mg, 0.00034 mole, 53%):mp 163°-166°; mass spectrum m/e 286 (M⁺), 271, 258, 243, 230, 215; νmax1730 (C═O) 1730cm⁻¹ (C═O); [θ] (MeOH) +4607° (305nm), -3016° (277nm),+1426° (248nm); nmr 0.93 (s,3,18-CH₃)

Anal. Calcd. for C₁₈ H₃₃ O₃ : C,75.49; H,7.74; O,16.77. Found: C,75.25;H,7.61; O,16.67.

EXAMPLE 30

3β,10β,17β-trihydroxy-5α-estran-6-one 3β,17β-Diacetate (9)

A mixture of 3β,17β-Diacetoxy-5β,10β-oxidoestran-6-one (4b, 800mg,0.0021 mole) and chromous acetate¹⁵ (3.4g, ca. 0.2 mole) was stirred atambient temperature for 24 hr in 90% aqueous acetone (150ml) under anatmosphere of argon. The reaction mixture was diluted with water andextracted with CHCl₃. The extract was washed with H₂ O, dried (Na₂ SO₄)and evaporated in vacuo. The residue was chromatographed on silica gel(elution with 11% EtOAc in CHCl₃). Early fractions gave unchangedoxidoketone (4b) followed by enone (3b). The most polar fractions gavethe desired β-hydroxyketone (9, 75mg, 0.00019 mole, 9%) mp 196°-199°;mass spectrum m/e 392 (M⁺), 376, 349, 314, 304, 272; νmax (KBr) 3480(OH), 1735cm⁻¹ (C═O); [θ] (CH₃ OH) -5078° (288nm).

Calcd. for C₂₂ H₃₂ O₆ : m/e 392.21900. Found: m/e 392.22510.

EXAMPLE 31

3β,20β-diacetoxy-19-norpregn-5(10)-en-6-one (3c)

Compound 3c was prepared in a manner identical with that for (3a) asdescribed above: mp 126°-127° (from acetone-light petroleum); massspectrum m/e 402 (M⁺), 342, 300, 254; λ_(max) ^(MeOH) 247nm (ε10,435);[θ] (MeOH) +2747° (330nm); νmax (CHCl₃) 1725 (ester C═O), 1660 (C═O),1625cm⁻¹ (C═C); nmr 4.82 (m,2,CHOAc), 2.0 (s,6,CH₃ CO), 0.65(s,3,18-CH₃)

Anal. Calcd. for C₂₄ H₃₄ O₅ : C,71.61; H,8.51. Found: C,71.60; H,8.26.

EXAMPLE 32 3β,20β-Diacetoxy-5β,10β-oxidopregnan-6-one (4c)

3β,20β-Diacetoxy-19-norpregn-5(10)-en-6-one (3c, 250 mg, 0.00062 mole)was dissolved in MeOH (18 ml) containing 30% H₂ O₂ (1.25 ml) and 5N NaOH(1.25 ml). After stirring 3 hr at ambient temperature the reaction wasdiluted with brine and extracted with CHCl₃. The extract was washed with5% NaHSO₃, dried with (Na₂ SO₄) and removed in vacuo. The residue wasacetylated (pyridine-acetic anhydride) and worked up in the usualmanner. Crystallization from hexane-EtOH afforded the pure oxide (4c,186 mg, 0.00045, 72%): mp 143°-145°; mass spectrum m/e 418 (M⁺), 360,358, 342, 340, 330, 298, 270; ν max 1740 (ester C═O), 1700 cm⁻¹ (C═O);[θ] (CH₃ OH) -9000° (307 nm); nmr 4.68 (m,2,CHOAc), 2.00 (s, 6,CH₃ CO),1.14 (d,3,J═6Hz, 21-CH₃), 0.68 (s,3,18-CH₃)

Anal. Calcd. for C₂₄ H₃₄ O₆ : C,68.87; H, 8.19, Found: C,68.71; H,8.11.

EXAMPLE 33

3β,20β-diacetoxy-5,10-seco-19-norpregn-5-yn-10-one (5c)

a mixture of 3β,20β-Diacetoxy-5β,10β-oxidopregnan-6-one (4c, 150 mg,0.00036 mole) and p-toluenesulfonylhydrazide (78 mg, 0.0043 mole) wasstirred for 6 hr. at ambient temperature in 1:1 CHCl₃ --AcOH (15 ml).The reaction mixture was diluted and extracted with CHCl₃. The extractwas washed successively with H₂ O, 5% NaHCO₃ and H₂ O. The residueobtained after drying (Na₂ SO₄) and removal of solvent was crystallizedfrom hexane-acetone to give 5c as plates (105 mg, 0.00026 mole, 72%): mp119°-120° mass spectrum m/e 418 (M⁺), 360, 358, 342, 340, 330, 298, 270;νmax 1735 cm⁻¹ (C═O); [θ] (CH₃ OH) -2098° (283 nm); nmr 4.80(m,2,CHOAc), 2.05 (s,3,CH₃ CO), 2.00 (s,3,CH₃ CO), 1.15(d,3,J═6Hz,21-CH₃),0.80 (s,3,18-CH₃)

Anal. Calcd. for C₂₄ H₃₄ O₆ : C,71.61; H,8.51, Found: C,71.53; H,8.64.

EXAMPLE 34

3β,20β-dihydroxy-5,10-seco-19-norpregn-5-yn-10-one (6c)

The diacetate (5c, 30 mg, 0.000075 mole) was refluxed in 3% methanolicKOH (7 ml) for 2 hr. The reaction was diluted with brine, extracted withCHCl₃, dried (Na₂ SO₄) and concentrated in vacuo to give 6c, (23 mg,0.00072 mole, 96%): mp 180°-182° (from hexane-EtOH) mass spectrum m/e318 (M⁺), 300, 285, 272; νmax (KBr) 3500 (OH), 1720 cm⁻¹ (C═O); [θ](MeOH) -3067° (283 nm); nmr 3.74 (m,2,CHOH), 1.09 (d,3,21-CH3), 0.83(s,3,18-CH₃)

Anal. Calcd. for C₂₀ H₃₀ O₃ : C,75.43; H,9.50, Found: C,75.38; H,9.50.

EXAMPLE 35

5,10-seco-19-norpregn-5-ny-3,10,20-trione (1c)

3β,20β-Dihydroxy-5,10-seco-19-norpregn-5-yn-10-one (6c, 140 mg, 0.00044mole) was dissolved in acetone (50 ml). Excess Jones reagent was addedand the mixture was stirred 5 min at ambient temperature. The residueobtained after dilution with water and extraction with CHCl₃ wascrystallized from hexane-EtOH to give 1c (75 mg, 0.00024, 54%): mp156°-159°; mass spectrum m/e 314 (M⁺), 286, 271; νmax (KBr) 1705 cm⁻¹ ;[θ] (dioxane) +4737° (301 nm); nmr 2.14 (s,3,21-CH₃ CO), 0.75(s,3,18-CH₃)

Anal. Calcd. for C₂₀ H₂₆ O₄ : C,76.40; H,8.34; O,15.66, Found: C,76.22;H,8.21; O,15.97.

In the preceding examples, it should be noted that melting points weredetermined on a Kofler hot stage and are uncorrected. Nmr spectra weredetermined on Varian HA-100 or Perkin-Elmer R-12B spectrometers forCDCl₃ solutions, unless otherwise stated, with TMS as internal standard.Chemical shifts are expressed as values (TMS = O) with signalmultiplicities shown as s, singlet; d, doublet; t, triplet; m,multiplet. Infrared spectra were obtained on Perkin-Elmer 137 or 521spectrometers (in CHCl₃ solution unless otherwise stated). Ultravioletspectra were measured on a Cary 15 spectrophotometer. Mass spectra weredetermined on CEC-21-110 or Dupont 21-491 spectrometers. Circulardichroism measurements were made using a Cary 60 instrument and areexpressed as molar ellipticities. All chromatographic separations wereperformed on Woelm dry column silica gal or alumina. Analyticalthin-layer plates (0.25 mm) were obtained from Analtech, Inc., Newark,Delaware. High pressure liquid chromatographic separations wereperformed on a Waters Associates Model 600 instrument equipped with aModel 660 solvent programmer.

The following data illustrates the inhibiting effect of estryne andpregnyne on Δ⁵ -3-ketosteroid isomerase (EC 5.3.3.1) as evidenced by adepression of the wet weight of the sex accessory tissue in the maturemale rat treated with the estryne and pregnyne.

The animals used in these tests were male Sprague-Dawley rate (250-300g); female, 21 day old CF-1 mice (12-15 g); and hypophysectomized maleSprague-Dawley rats (200-250 g). The animals were housed in atemperature controlled room under a constant lighting schedule andmaintained on a diet of Charles River Rat Formula and water ad libitum.The diet of the hypophy-sectomized animals was supplemented withorganges.

Study in Intact Rats

A. Male rats were used to assess the effect of estryne on endogenousandrogen biosynthesis at varying doses (1-20 mg/kg body weight). Theestryne was administered interperitoneally in propylene glycol (0.2 ml)daily for 7 days. On the eighth day the animals were sacrificed and theventral prostate, dorsal lateral prostate, seminal vesicles, testes,kidneys, and adrenals were removed and weighed. Samples of each tissuewere preserved in 10% buffered formalin for histological examination.

B. Male rats were treated with 19-norpregnyne by a regimen exactly asdescribed for the estryne.

C. To assess the effect of body weight decrease on the wet weight of sexaccessory tissue, animals were deprived of food while receiving water adlibitum until they lost 20% body weight relative to controls, and weresacrificed on the appropriate day for organ weight determination.

Study in Castrate Rats

To determine if the estryne competes with testosterone at the targettissue rats were orchiectomized under ether anesthesia via the scrotalroute 10 days prior to treatment. Testosterone propionate 0.3 mg (0.2 mlsesame oil) was administered daily for 7 days subcutaneously. Estryne 12mg/kg (0.2 ml propylene glycol) was given daily for 7 daysinterperitioneally. Control groups received either testosteronepropionate (0.3 mg) or carrier oil daily for 7 days. The animals weresacrificed on the eighth day and the organs were weighed.

Study in Hypophysectomized Rats

In order to assess the action of the estryne in the presence ofexogenous stimulation of androgen synthesis, hypophysectomized male ratsreceived estryne 3.0 mg (0.2 ml sesame oil) sc and/or human chorionicgonadotropin 2 units and 4 units (0.9% saline) sc daily for 7 days.Sacrifice and weighing of the organs was performed on the eighth day.

Study of Estrogenic Activity

Estrogenic activity was determined in 21 day old CF-1 female micereceiving either estryne (1-100 μg) or estradiol (4 mg-192 mg) b.i.d. ×3 days in 10% dioxanesesame oil (0.05 ml) sc. On the fourth day theuteri were removed and weighed.

Study of the Binding to the Prostate Androgen Binding Protein

The binding to the cytosol androgen receptor was determined by theprocedure of Fang (5) with modification. Thus, 400 mg of freshly mincedventral prostate tissue obtained from rats castrated 24 hours beforesacrifice was incubated at 37° for 1 hour in 5 ml of TM buffer (50 mMtris, 3 mM Mg Cl₂, 0.3 M sucrose) pH 7.4 containing 14 μCi of ³H-dihydrotestosterone (85 Ci/mM, New England Nuclear Corp., Boston,Mass.), together with estryne or pregnyne (2 × 10⁻⁶ M) under anatmosphere of 95% oxygen and 5% carbon dioxide. Cyproterone acetate andunlabelled dihydrotestosterone were used as steroidal controls.Following incubation the tissue was washed three times with cold TM (5ml) and the nuclei were isolated by the method of Coffey et al. (Coffey,D. S. Shimazaki, J. and Williams-Ashman, H. G., Arch. Biochem. Biophys.,124, 184 (1968). The amount of radioactivity in the nuclei is expressedas observed counts per minute per 100 μg DNA. The amount of DNA wasassayed by the Burton diphenylamine method, with calf thymus DNA as thereference (see Burton, K., Methods in Enzymology, vol. 12B, 163, L.Crossman and K. Maldane (eds.), Academic Press, New York, 1968).

Results

Since growth inhibition or cell death of the sex accessory tissue canresult from multiple mechanisms, it was necessary to study the mode ofaction of the estryne and 19-nor-pregnyne. The following modes of actionwere considered: (a) direct competition with testosterone anddihydrotestosterone for the androgen nuclear binding protein of theventral prostate, (b) suppression of luteinizing hormone production byaction on the hypothalamus or pituitary, (c) direct estrogenic action,(d) inhibition of androgen biosynthesis.

Studies in Intact Rats

A. The acetylenic steroid analogue 5,10-secoestr-5-yne-3,10,17-trione(estryne) depresses the wet weight of both the ventral anddorsal-lateral prostate in short term treatment (7 days). Only a modestdecrease is observed for the seminal vesicles, and no effect on thetestes,or kidney, or adrenal for the same treatment. The estryne causeda decrease (25-30%) in the wet weight of both the ventral anddorsal-lateral prostate at a daily dose (7 days) of 20mg/kg/body weight.The effect on the prostatic weight was dose dependent for the estryne.(Table 1).

B. The 19-norpregnyne analogue decreased the weight of the ventral anddorsal-lateral prostate by the same percent as the estryne at a dose of20mg/kg/body weight for 7 days. The weight decrease was also dosedependent (Table 2).

C. Both acetylenic steroids were equally effective at a given doseadministered intraperitoneally or subcutaneously. At a dose of20mg/kg/body weight for 7 days by the IP route a 20% decrease in bodyweight relative to controls occurred. This loss was not observed forsubcutaneous injection or lower doses. In order to access the possibleeffect of weight loss on the prostate, animals were deprived of fooduntil they had lost 20% body weight relative to controls. No significantchange in prostatic weight was observed.

Study in Castrate Rats

Animals castrated 10 days prior to treatment received both estryne(12mg/kg weight) and testosterone propionate (0.3mg) daily for sevendays. No significant depression of prostatic weight was observed. Thissuggests that the estryne is not blocking the action of androgen at thetarget tissue by preventing dihydrotestosterone from entering thenucleus.

Study in Hypophysectomized Rats

Since the estryne does not appear to inhibit the action of androgens atthe target tissue in vivo it was necessary to determine if it couldantagonize the effect of human chorionic gonadotropin (HGG) whichstimulates androgen biosynthesis in the testes of hypophysectomizedrats. The wet weight of the ventral prostate was decreased (20-25%) inhypophysectomized animal receiving estryne (12mg/kg body weight) and HGG(2 units) as compared to controls receiving just HGG (2 units). Theeffect was less pronounced at higher doses of HGG.

estrogenic Activity

Due to the potency of estrogens and their marked effect on androgenbiosynthesis via the gonadal-pituitary axis the estryne was assayed forestrogenic activity. Thus, immature female mice received estryne atdoses up to a maximum of 100 μg total dose over three days, and the wetweight of the uteri were determined. A 100 μg total dose, the estryneshowed no stimulation of the immature uterus whereas a dose as low as0.67 μg of 17β-estradiol gave a positive response, with maximumstimulation at about 8 μg using the same dosage schedule as for theestryne.

Study of Binding to the Prostatic Androgen Receptor

A. Incubation of estryne (2 × 10⁻⁶ M) with rat ventral prostate micesinhibited the nuclear uptake of ³ H-dihydrohydrotestosterone (2 × 10⁻⁹M, 85 Ci/mM) by approximately 50% as determined by the radioactivitypresent in the isolated nuclei (Table 3).

B. Interestingly incubation of 19-norpregnyne with ventral prostateminces as above showed no inhibition of androgen uptake by the nuclei(Table 3).

                                      Table 1                                     __________________________________________________________________________    TREATMENT OF MATURE INTACT RATS WITH ESTRYNE FOR SEVEN DAYS                   __________________________________________________________________________                    Dorsal                                                                  Ventral                                                                             Lateral                                                                             Seminal                                                 Daily Dose                                                                              Prostate                                                                            Prostate                                                                            Vesicles                                                                             Adrenal                                                                             Testis Kidney                              __________________________________________________________________________                    (mg ± Sem)                                                 Intact Control                                                                          421 ± 15                                                                         244 ± 12                                                                         394 ± 12                                                                          27 ± 2                                                                           1650 ± 38                                                                         1260 ± 63                        Castrate Control                                                                         76 ± 7                                                                          111 ± 9                                                                          249 ± 20                                                                          28 ± 1                                                                           --     1220 ± 33                         1 mg/kg  435 ± 23                                                                         212 ± 19                                                                         380 ± 18                                                                          28 ± 2                                                                           1650 ± 60                                                                         1320 ± 68                         5 mg/kg  385 ± 8                                                                          195 ± 8                                                                          368 ± 16                                                                          26 ± 2                                                                           1590 ± 54                                                                         1090 ± 39                        10 mg/kg  364 ± 25                                                                         188 ± 6                                                                          387 ± 14                                                                          30 ± 2                                                                           1580 ± 59                                                                         1150 ± 42                        20 mg/kg  302 ± 28                                                                         177 ± 15                                                                         309 ± 23                                                                          30 ± 3                                                                           1420 ±  64                                                                        1040 ± 35                        __________________________________________________________________________

                                      Table 2                                     __________________________________________________________________________    TREATMENT OF MATURE INTACT RATS WITH PREGNYNE FOR SEVEN                       __________________________________________________________________________    DAYS                                                                                          Dorsal                                                                  Ventral                                                                             Lateral                                                                             Seminal                                                 Daily Dose                                                                              Prostate                                                                            Prostate                                                                            Vesicles                                                                             Adrenal                                                                             Testis Kidney                              __________________________________________________________________________                          (mg ± Sem)                                           Control   482 ± 24                                                                         252 ± 14                                                                         470 ± 26                                                                          28 ± 2                                                                           1580 ± 23                                                                         1410 ± 38                         1 mg/kg  405 ± 18                                                                         205 ± 14                                                                         423 ± 21                                                                          22 ± 2                                                                           1530 ± 68                                                                         1360 ± 48                         5 mg/kg  402 ± 27                                                                         208 ± 11                                                                         384 ± 17                                                                          25 ± 2                                                                           1510 ± 44                                                                         1370 ± 42                        10 mg/kg  386 ± 20                                                                         182 ± 19                                                                         390 ± 14                                                                          26 ± 1                                                                           1530 ± 53                                                                         1310 ± 60                        20 mg/kg  360 ± 21                                                                         180 ± 14                                                                         380 ± 9                                                                           31 ± 2                                                                           1480 ± 63                                                                         1360 ± 43                        __________________________________________________________________________

                  Table 3                                                         ______________________________________                                        Inhibition of Nuclear Uptake of .sup.3 H-Dihydrolestosterone (DHT)            by Estryne and Pregnyne in the Rat Ventral Prostate                           ______________________________________                                               Conc     μ g    CPM/100                                             Steroids                                                                             (M)      DNA/ml    DNA       % Control                                 ______________________________________                                        .sup.3 H-DHT                                                                         2 × 10.sup.-9                                                                    237       8,471     100                                       (Control)                                                                     DHT    2 × 10.sup.-6                                                                    255       117        1.4                                      .sup.3 H-DHT                                                                         2 × 10.sup.-9                                                    Cp-Ac* 2 × 10.sup.-6                                                                    196       941        11.1                                     .sup.3 H-DHT                                                                         2 × 10.sup.-9                                                    Estryne                                                                              2 × 10.sup.-6                                                                    197       4,771      56                                       .sup.3 H-DHT                                                                         2 × 10.sup.-9                                                    Pregnyne                                                                             2 × 10.sup.-6                                                                    230       11,636    137                                       .sup.3 H-DHT                                                                         2 × 10.sup.9                                                     ______________________________________                                         Cyproterone Acetate?                                                     

As noted above, the acetylenic compounds of the invention, e.g. estryneand pregnyne, are converted into the corresponding conjugated allenicketones which then react irreversibly with the enzyme. These allenicketones are also novel and constitute an important embodiment of theinvention. The allenic ketones derived from estryne are6β(H)-5,10-secoestra-4,5-diene-3,10,17-trione and6α(H)-5,10-secoestra-4,5-diene-3,10,17-trione. These allenic ketoneshave been tested as inhibitors of isomerase and have been foundeffective in this regard. Under pseudo first order conditions(isomerase, 4,80 (μM; allenic ketone, 200 μM; 1 mM phosphate buffer, pH= 7, 6β(H)-5,10-secoestra-4,5-diene-2,10,17-trione is partiallyconverted to the 6α isomer and a half life of 540 sec for loss of enzymeactivity was observed. Likewise the 6α allenic compound was partiallyconverted to the β-isomer and gave a half life of 660 sec for loss ofenzyme activity. The allenic ketones derived from pregnyne give similarresults.

The following examples further illustrate preparation of the allenicsteroids of the invention and their utility.

EXAMPLE 36

5,10-seco-19-norpregna-4,5-diene-3,10,20-trione

5,10-seco-19-norpregn-5-yne-3,10,20-trione (200mg, 0.64 mmol) andtriethylamine (363mg, 3.59 mmol) were stirred in dioxane (20ml) at roomtemperature for 3 hr. The solvent and base were removed under oil pumpvacuum and the residue chromatographed on dry column silica gel with a5:1 hexane/acetone mixture as eluent. High pressure liquidchromatography was used to analyze the column fractions. Aftercombination of fractions there resulted, 39mg (20%) of6β(H)-5,10-seco-19-norpregna-4,5-diene-3,10,20-trione, mp 122°-124°,ν_(max) ^(CHCl).sbsp.3 1940, 1700, 1665 cm⁻¹. An additional mixture(138mg; 69%) which contained the 6β(H)- and 6α(H)-allenes in the ratio85:15 was also obtained. A portion of this mixture was subjected toHPLC, giving 6α(H)-5,10-seco-19-norpregna-4,5-diene-3,10,20-trione,ν_(max) ^(CHCl).sbsp.3 1940, 1700 and 1665 cm⁻¹.

EXAMPLE 37

5,10-secoestra-4,5-diene-3,10,17-trione

5,10-secoestr-5-yne-3,10,17-trione (200mg, 0.70 mmol) and triethylamine(363mg, 3.59 mmol) were stirred in dioxane (20ml) at room temperaturefor 3 hr. The solvent and base were removed under aspitator vacuum on arotary evaporator and the residue chromatographed on dry column silicagel with a 5:1 hexane/acetone mixture as eluent. High pressure liquidchromatography was used to analyze the column fractions. Aftercombination of fractions, 79mg (39%) of6β(H)-5,10-secoestra-4,5-diene-3,10,17-trione was obtained, mp 144°,ν_(max) ^(CHCl).sbsp.3 1945, 1735, 1710 and 1680 cm⁻¹. An additionalmixture (80mg. 40%) which contained the 6β(H)- and 6α(H)-allenes in a76:24 ratio was also obtained. A portion of this mixture was subjectedto HPLC, giving 6α(H)-5,10-seco-19-norestra-4,5-diene-3,10,7-trione,ν_(max) ^(CHCl).sbsp.3 1965, 1740, 1710, 1675 cm⁻¹.

EXAMPLE 38

Effects of 6β(H)-5,10-Seco-19-norestra-4,5-diene-3,10,17-trione onintact male Sprague-Dawley rats

The compound (0.6mg) dissolved in propylene glycol (0.2ml) wasadministered daily for 7 days by the intraperitoneal route. Theexperimental group and control group each comprised 5 animals; andweghts of animals as well the wet tissue weights of ventral prostate anddorsal lateral prostate are averaged for each group.

    ______________________________________                                                         Wt. of                                                                 Wt. of Ventral  Wt. of Dorsal                                                 Animals                                                                              Prostate Lateral Prostate                                    ______________________________________                                        Experimental Group                                                                        382 g.   488 mg.  235 mg.                                         Control Group                                                                             388 g.   574 mg.  299 mg.                                         ______________________________________                                    

It will be appreciated that various modifications may be made in theinvention described herein. Hence the scope of the invention is definedby the following claims wherein:

I claim:
 1. An allenic steroid selected from the group of formulasconsisting of: ##STR12## wherein R¹ is CH₂ ═ , O ═ or ##STR13## R² is O═, ##STR14## (where R³ is H or lower alkanoyl); ##STR15## (where R⁴ is Hor lower alkanoyl); ##STR16## (where R⁵ is lower alkyl) or ##STR17##(where R⁶ is H, lower alkyl or Cl); and R⁷ is H or CH₃. 2.6β(H)-5,10-secoestra-4,5-diene-3,10,17-trione. 3.6α(H)-5,10-secoestra-4,5-diene-3,10,17-trione.
 4. 6β(H)-5,10-secopregna-4,5-diene-3,10,20-trione.
 5. 6α(H)-5,10-secopregna-4,5-diene-3,10,20-trione.